Triapine (3-AP), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of ER stress in the anticancer activity of 3-AP and the derivative 3-AP-Me, differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a and ATF6) by phosphorylation of eIF2α and upregulation of ATF4 and ATF6 gene expression, and particularly 3-AP-Me lead to an upregulation of the alternatively spliced mRNA variant XBP1s (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases JNK and p38 MAPK, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response (UPR), a significant upregulation of pro-apoptotic proteins was detected, including the transcription factor CHOP and the BH3-only member protein Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the anti-proliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal methylation leads to an even more pronounced manifestation of this effect.